In all cells, the site of protein synthesis is the ribosome. This complex ribonucleoprotein particle transiently binds all the major participants in the process, and directs the efficient and accurate synthesis of protein molecules from the genetic blueprint. The importance of a correctly functioning ribosome to the cell is reflected in the many drugs and antibiotics that are produced to disrupt its operation in cells targeted for destruction. Relatively little is known about how the ribosome operates at the molecular level. During the past five years, we have determined the structures of several bacterial ribosomal proteins and have gained extensive insight into the structure, mechanism and evolution of the particle. It is proposed to continue this work during the next five years, and to expand its scope to include protein-RNA interactions and ribosome-associated factors. The ability to clone any bacterial ribosomal protein gene means that components which are particularly crucial to the ribosome can be selected for analysis. The proteins that have been chosen for the next grant period are S7, S8 and S17 which control the initial folding of the RNA component, and S4 and S12 which mediate the ribosome's accuracy. In addition, the structures of two factors, IF2 and IF3, that are essential for the initiation of protein synthesis will be determined. The previous structural analyses were greatly facilitated by working with the highly stable proteins from thermophilic organisms, and this will continue. The genes for each of the chosen ribosomal components have been cloned from various thermophilic bacteria, and considerable progress has already been made towards the elucidation of their structures. Finally, in vitro selection techniques will be used to investigate the RNA-binding sites of selected proteins. The aim of this work will be to form a defined protein- RNA complex for structural studies. The principal technique for our structural research has been X-ray crystallography using both conventional X-ray sources and synchrotron radiation. This will continue to be the case, but extensive use will also be made of NMR methods during the next five years. The majority of the proteins that will be studied are within the size limits of NMR, and the preliminary structure of one protein, S17, has already been determined using this technique. Finally, this work has attracted great interest within the ribosome research community. Several collaborations have developed during the past five years with groups interested in the biochemistry and molecular biology of the ribosome, and these will be developed and expanded.